| Cough variant asthma is a special type of asthma and also a common disease of respiratory system.Its main clinical manifestations were cough,unconsp-icuous wheezing,and shortness of breath,but airway hyperresponsiveness.In recent years,the incidence of CVA is increasing significantly,which has a great impact on patients’ work and life.It is believed that western medicine has a rapid and clear effect in the treatment of CVA,but there is a large side effect,easy to relapse and other problems,while traditional Chinese medicine is treat-ing both manifestation and root cause of disease,and the recurrence rate is low.Therefore,it is very important to study the traditional Chinese medicine for treating CVA and explore its mechanism.Zihepochuan Particles is experience prescription of Professor Li Youlin of China-Japan Friendship Hospital,mainly for the treatment of CVA.It is composed of astragalus root,cassia twig,liquo-rice,aster,Magnolia,Terminalia ten Chinese medicinal herbs,and the main ef-fect is invigorating the spleen and benefiting the lung and relieving cough and asthma.Previous clinical studies have shown that Zihepochuan Particles has a significant therapeutic effect on CVA,and can significantly reduce the level of IgE.Studies have shown that the pathogenesis of CVA is related to the imbal-ance of Th1/Th2/Th17 cytokines,while leukotriene is an important inflammatory mediator which caused CVA.Based on that,this study established the CVA mouse model and rat model induced by OVA,and observed its effect on the treatment of CVA and the effect on the 5-LOX/LTs/CysLTR signaling pathway,which provide reference for its further application.1 Effect of Zihepochuan Particles on mice with CVA induced by OVAObjective:To research the effects of Zihepochuan Particles on IgE,expression of inflammatory cytokine and the effects of lung histopathology of model of mice with CVA.Methods:SPF grade BALB/c mice were randomly divided into 6 groups:normal group,model group and prednisone acetate group(0.004 g/kg),Zihepochuan Particles of high-dose group(10.66 g/kg),middle-dose group(5.33 g/kg)and low-dose group(2.67 g/kg).In addition to the normal group,other groups were injected multipoint subcutaneously and intraperitoneally with OVA and Al(OH)3.Repeat first days of operation after 1 weeks.The mice in the normal group were given the same method of injection of the same dose of saline.The drug is given next day.7 days after administration,the serum TNF-alpha and IFN-gamma,IgE,IL-4,IL-10 and IL-17 were measured by enzyme linked immunosorbent assay(ELISA),and the pathological of lung tissue were observed under light microscope after HE staining.Results:IgE,expression of Inflammatory cytokine:Compared with the normal group,the serum levels of IgE,IL-4 and IL-17 significantly increased(P<0.05 or 0.01)in the model group of mice,the level of IFN-y significantly reduced(P<0.05),and the level of IL-10 and TNF-α was no obvious changed(P>0.05).Compared with model group,the level of IgE was significantly recuded(P<0.05 or 0.01)in the prednisone acetate group,Zihepochuan Particles of high-dose and middle-dose gourp,the level of IL-4 significantly recuded(P<0.05)in prednisone acetate group and Zihepochuan Particles of high-dose group,the level of IL-17 significantly reduced(P<0.05)in prednisone acetate group,Zihepochuan Particles of high-dose and low-dose group,the level of IFN-y increased(P<0.05)in prednisone acetate group,Zihepochuan Particles of middle-dose group,the level of IL-10 and TNF-α was no obvious changed.(P>0.05).Pathological changes of lung tissue:The structure of each layer of bronchi in the lung tissue of CVA mice was severely damaged.The necrosis and exfoliation of the bronchial epithelium,the loose surrounding tissue of the pulmonary vessels and infiltration of inflammatory cells were found.Zihepochuan Particles can protect all layers of bronchus,and the mucosal epithelium is less damaged.Occasionally,inflammatory cells infiltrate the bronchial wall.Conclusion:The results shows that Zihepochuan Particles can recude the level of IgE,IL-4 and IL-17,improve the level of IFN-γ in serum of CVA mice,and reduce lung injury.2 Effect of Zihepochuan Particles on rats with CVA induced by OVAObjective To research the effects of Zihepochuan Particles on airway resistance,IgE,expression of Inflammatory cytokine and and histopathology of lung tissue of CVA rats.Methods SPF BN rats,were randomly divided into seven groups:normal group,model group,montelukast sodium group(0.0013 g/kg),prednisone acetate group(0.004 g/kg),high dose group(10.66 g/kg),middle dose group(5.33 g/kg)and low dose group(2.67 g/kg).CVA rat model was prepared by intraperitoneal injection of OVA.On the first day,OVA and Al(OH)3 suspension were injected intraperitoneally.Repeat after 3 weeks,and the normal group is replaced by equal amount of saline.After 3 weeks,the drug was given.After 14 days,methacholine test was used to detect airway resistance in rats,the serum contents of IgE,IL-4,IL-5,IL-6,IL-13,IL-17 and IFN-y were determined by ELISA,lung tissue pathological morphological was observed under light microscope after HE staining and scored.Results:Airway Resistance:Compared with normal group,the airway resistance ofrats in the model group significantly increased in the four concentrations of methacholine(P<0.05 or 0.01).Compared with model group,the airway resistance of rats in the montelukast sodium group significantly decreased in concentration of methacholine in 0.05mg/kg(P<0.05),the airway resistance significantly decreased in the prednisone acetate group in concentration of methacholine in 0.05mg/kg and 0.1mg/kg(P<0.05),and the middle-dose group was significantly decreased in the concentration of methacholine in 0.05mg/kg and 0.2mg/kg(P<0.05).IgE,expression of Inflammatory cytokine:Compared with the normal group,the serum levels of IgE,IL-5 IL-13 and IL-17 was significantly increased(P<0.05 or 0.01)in the model group,the level of IFN-y significantly decreased(P<0.05),and the level of IL-4 and IL-6 was no obvious changed(P>0.05).Compared with model group,the level of IgE was significantly recuded(P<0.05 or 0.01)in montelukast sodium group,prednisone acetate group and middle dose gourp,the level of IL-5 significantly recuded in montelukast sodium group(P<0.05),the level of IL-6 significantly increased in prednisone acetate group(P<0.05),the level of IL-17 significantly recuded(P<0.05),the level of IFN-y significantly increased in prednisone acetate group(P<0.01),the level of IL-4 significantly increased in high dose and low dose group(P<0.05),and the level of IL-13 was no obvious changed(P>0.05).Pathological changes of ung tissue:Compared with normal group,the airway secretions were increased,inflammatory cells were infiltrated,and thepathological score was significantly higher(P<0.01)in the model groups.Compared with model group,in high dose group,the airway secretions were greatly reduced and the inflammatory cell infiltration was rate,and the pathological score decreased significantlyrely(P<0.01),and in middle-dose and low-dose group,the airway secretions were reduced,and the inflammatory cell infiltration was severe,and the pathological score decreased significantly(P<0.05 or 0.01).Conclusion:Zihepochuan Particles can reduce airway resistance,sercum level of IgE,IL-17,increase IL-4,and reduce lung injury.3 Study on action mechanism of 5-LOX/LTs/CysLTR signaling pathway of Zihepochuan ParticlesObjective:To observe the effect of Zihepochuan Particles on the 5-LOX/LTs/CysLTR signaling pathway,and explore the possible mechanism for the treatment of CVA.Methods:The CVA rat model was established by the same method as experiment two.The serum contents of 5-LOX、FLAP、LTB4、LTC4 and LTD4 were determined by ELISA,immunohistochemical staining of 5-LOX in lung tissue,detection of mRNA expression of CysLT1 receptor in lung tissue by real-time fluorescence quantitative PCR,the expression of CysLT1 protein in lung tissue was detected by Western-blot.Results:Biochemical indices:Compared with the normal group,the serum level of 5-LOX,FALP,LTB4 and LTD4 was significanty increased in model group(P<0.05 or 0.01)and LTC4 was no obvious changed(P>0.05).Compared with model group,the level of 5-LOX was significanty reduced in high dose group(P<0.01),the level of FLAP significanty reduced in prednisone acetate group and high dose group(P<0.05 or 0.01),the level of LTB4 significanty reduced in middle dose group(P<0.01),the levelof LTC4 significanty reduced in montelukast sodium group and high dose group(P<0.05 or 0.01),and the level of LTD4 was no obvious changed(P>0.05).Immunohistochemical staining:Compared with normal group,the masculine cell counting of 5-LOX in the the lung tissue was significantly increased(P<0.05).Compared with model group,the masculine cell counting significantly reduced in all drug delivery groups(P<0.05 or 0.01).Real time fluorescence quantitative PCR:Compared with normal group,the expression of CysLT1 mRNA in lung tissue of model group was significantly increased(P<0.01).Compared with model group,the expression of CysLT1 mRNA was significantly reduced significantly in montelukast sodium group,prednisone acetate group,middle-dose and low-dose group(P<0.05 or 0.01).Western-blot:Compared with normal group,the expression of CysLT1 receptor protein was significantly increased(P<0.05).Compared with model group,the expression of CysLT1 receptor protein significantly decreased in all drug delivery groups(P<0.01).Conclusion:Zihepochuan Particles can reduce serum level of 5-LOX,FLAP,LTB4,LTC4,and the expression of 5-LOX CysLT1 mRNA and protein in lung tissue.The above results suggest that it can reduce the airway resistance,regulate the expression of related cytokines,and alleviate the pathological damage of lung tissue of CVA animal models.The link may be related to imbalance of Thl/Th2/Th17 cytokine and 5-LOX/LTs/CysLTR signaling pathway. |
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